Establishing an itaconic acid production process with Ustilago species on the low-cost substrate starch

Abstract Ustilago maydis and Ustilago cynodontis are natural producers of a broad range of valuable molecules including itaconate, malate, glycolipids, and triacylglycerols. Both Ustilago species are insensitive toward medium impurities, and have previously been engineered for efficient itaconate production and stabilized yeast-like growth. Due to these features, these strains were already successfully used for the production of itaconate from different alternative feedstocks such as molasses, thick juice, and crude glycerol. Here, we analyzed the amylolytic capabilities of Ustilago species for metabolization of starch, a highly abundant and low-cost polymeric carbohydrate widely utilized as a substrate in several biotechnological processes. Ustilago cynodontis was found to utilize gelatinized potato starch for both growth and itaconate production, confirming the presence of extracellular amylolytic enzymes in Ustilago species. Starch was rapidly degraded by U. cynodontis, even though no α-amylase was detected. Further experiments indicate that starch hydrolysis is caused by the synergistic action of glucoamylase and α-glucosidase enzymes. The enzymes showed a maximum activity of around 0.5 U ml−1 at the fifth day after inoculation, and also released glucose from additional substrates, highlighting potential broader applications. In contrast to U. cynodontis, U. maydis showed no growth on starch accompanied with no detectable amylolytic activity.


Introduction
In view of the gro wing w orld population and the ov er exploitation of fossil fuels, a tr ansition fr om the fossil-based to bio-based production of chemicals from renewable resources is indispensable (Stegmann et al. 2020 ).This has already been successfully done for different carboxylic acids like citric , lactic , succinic , or itaconic acid (Chen andNielsen 2016 , Kuenz andKrull 2018 ).Itaconic acid belongs to the 12 most promising bio-based platform chemicals defined by the U.S. Department of Energy in 2004 (Werpy and Petersen 2004 ).It is of particular interest as an alternative for petr oc hemical-based acrylic and methacrylic acid in the polymer industry (Teleky and Vodnar 2019 ), but also has a variety of biological activities that makes it r ele v ant in medical and pharma-ceutical sectors (Michelucci et al. 2013, Mills et al. 2018, Olagnier et al. 2020 ).
To date, itaconic acid is commerciall y pr oduced using Aspergillus terreus ac hie ving titers up to 160 g l −1 and yields of up to 0.58 g ITA g GLC −1 in pulsed batch fermentations on glucose (Krull et al. 2017 ).Ho w e v er, to be competitiv e with the petr oc hemical sector, production costs need to be further reduced, which are significantly influenced by the feedstock used (Saur et al. 2023 ).
One alternative feedstock offering potential cost reductions is the pol ysacc haride starc h.Starc h is highly abundant in nature and the most widely utilized substrate for biofuel production.It can be obtained from a variety of agricultural raw materials such as potatoes, wheat, and corn for industrial production in r elativ el y high purity, simplifying downstr eam pr ocessing (Celi ńska et al. 2021, Singh et al. 2022 ).Its metabolization entails the liquefaction by an α-amylase and subsequent saccharification into glucose by a glucoamylase (Ebrahimian et al. 2022 ).Since the increased cultiv ation of starc h-containing plants for industrial pur poses can be negativ el y perceiv ed in public opinion, there is a growing interest in utilizing starchy side str eams fr om food pr ocessing industry (Jagadeesan et al. 2020, Kumar et al. 2023, Rodriguez-Martinez et al. 2023 ).The usa ge of suc h industrial side-and waste str eams not onl y r educes pr oduction costs, it also makes it possible to ac hie v e the circular bioeconom y conce pt without compromising food security (Leong et al. 2021 ).Since A. terreus is highly sensitive to medium impurities, the use of such more complex substr ates r equir es pr etr eatment in order to r emov e tr ace elements, which is in addition to the laborious handling and difficult oxygenation due to its filamentous growth one major dr awbac k of this pr oduction or ganism (Klement and Büc hs 2013 ).Ther efor e, we focus on the basidiomycetes Ustilago maydis and Ustilago cynodontis as alternative natural itaconate producing strains.Both strains hav e alr eady been metabolicall y and mor phologicall y engineer ed to maintain yeast-like growth and to enable high-le v el itaconate production at the maximum theoretical yield of 0.72 ± 0.02 g ITA g GLC −1 in the production phase with a constant glucose feed (Hosseinpour Tehrani et al. 2019a,b , Becker et al. 2021 ).The robustness of Ustilago species to medium impurities and its r epertoir e of hydr ol ytic, secr etory enzymes makes it a promising candidate for itaconate production based on more complex substrate in a consolidated process (Mueller et al. 2008, Becker et al. 2023 ).This has alr eady been demonstr ated by the use of the untr eated, sucr osecontaining side streams molasses and thick juice from sugar industry as well as crude gl ycer ol fr om biodiesel pr oduction as feedstock for Ustilago -based itaconate production (Helm et al. 2023, Niehoff et al. 2023, Saur et al. 2023 ).Furthermor e, activ ation of intrinsic xylanases , cellulases , and pectinases enabled degradation of the plant cell wall components hemicellulose , cellulose , and pectin (Geiser et al. 2016, Müller et al. 2018, Stoffels et al. 2020 ), e v en though direct usage of lignocellulosic biomass usually r equir es costl y pr etr eatment to destr oy its r ecalcitr ant structur e (Regestein et al. 2018 ).
Here, we performed a proof-of-concept study on the amylolytic potential of U. maydis and U. cynodontis for the direct utilization of potato starch as a feedstock for itaconate production in a consolidated bioprocess.

Chemical and strains
The chemicals used in this study were obtained from Sigma-Aldrich (St. Louis , USA), T hermo Fisher Scientific (Waltham, USA), or VWR (Radnor, USA) and were of analytical grade.
All strains used in this work are listed in Table 1 .
The vitamin solution contained (per liter) 0.05 g d -biotin, 1 g dcalcium pantothenate, 1 g nicotinic acid, 25 g myo-inositol, 1 g thiamine hydr oc hloride, 1 g pyridoxol hydr oc hloride, and 0.2 g par aaminobenzoic acid.The trace element solution contained (per liter) 1.5 g EDTA, 0.45 g of ZnSO   C, and = 80%).For growth and production experiments, main cultur es wer e inoculated to an OD 600 of 0.5 with pr ecultur es gr own in the same media.The DASGIP Bioblock system (Eppendorf, Germany) was used to conduct the batc h fermentations, whic h wer e contr olled using the Eppendorf DASwar e ® contr ol softwar e (Eppendorf, German y).
Vessels with a total volume of 2.3 l and a working volume of 1.0 l were used.The cultivations were performed in batch medium according to Geiser et al. ( 2014 ) as described abo ve .T he medium also contained 1 g l −1 yeast extract (Merck Millipore, Germany) and either 100 g l −1 gelatinized potato starch or 200 g l −1 α-amylase pr etr eated potato starch.To avoid clumping of starch during autoclaving, a slurry was pr epar ed befor ehand b y mixing star ch in hot water.For the α-amylase pr etr eatment, a 1% (v/v) solution of heat-stable Bacillus licheniformis α-amylase (Sigma-Aldrich) was added through the septum and incubated for ∼2 h.The slurry was maintained at 80 • C during the pr etr eatment.Afterw ar ds, the temper atur e was adjusted to 30 • C, and the remaining medium compounds were added through the septum.Finally, the bioreactor was inoculated to a OD 600 of 0.75 from an pr ecultur e gr own in screening medium according to Geiser et al. ( 2014 ) containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and 50 g l −1 gelatinized potato star ch.The pH w as automatically controlled by adding 5 M NaOH or 1 M HCl, while the DO was maintained at 30% using a cascade that involved agitation at 800-1200 rpm (0%-40% DOT controller output), air flow at 1-2 vvm (40%-80% DOT controller output), and oxygen at 21%-100% oxygen (80%-100% DOT controller output).Additionally, 0.5 ml of Antifoam 204 (Sigma-Aldrich) was added at the beginning of the cultivation and every 24 h thereafter.

Analytical methods
Identification and quantification of products and substrates present in the supernatants were conducted using a high performance liquid c hr omatogr a phy (HPLC) 1260 Infinity system (Agilent, Waldbronn, Germany) equipped with an ISERA Metab AAC column 300 mm × 7.8 mm column (ISERA, German y).Separ ation was ac hie v ed thr ough isocr atic elution at a flow r ate of 0.6 ml min −1 and a temper atur e of 40 • C, employing 5 mM sulfuric acid as a solv ent.Detection involv ed a diode arr ay detector at 210 nm and a r efr action index detector.Anal ytes wer e identified based on their retention time compared to corresponding authentic standards, and data analysis was performed using the Agilent OpenLAB Data Analysis-Build 2.200.0.528 software (Agilent).Ammonium concentrations in culture samples were determined using the colorimetric method outlined by Willis et al. ( 1996 ).In this method, 50 μl culture supernatant was mixed with 1 ml r ea gent (8 g sodium salicylate , 10 g trisodiumphosphate , and 0.125 g sodium nitroprusside), follo w ed b y r a pid addition of 250 μl hypoc hlorite solution.After color de v elopment, the absorbance was measured at 685 nm using cuvettes and a spectrophotometer.Ammonium concentrations were calculated using an ammonium calibration.Cell densities were quantified by measuring the optical density at a wavelength of 600 nm (OD 600 ) using cuvettes and a spectrophotometer.Samples were diluted appropriately with the respective medium to ensure measurement within the linear range of the photometer, falling between absolute values of 0.2 and 0.4.SDS-PAGE was performed according to the manufacturer's instructions using Nu-PAGE 12% Bis-Tris precast gels and MOPS as the running buffer.A volume of 10 μl of each sample and 5 μl of the protein ladder were loaded onto the gel.After electrophoresis, the gels were stained with Coomassie (Gel Code Blue).For the detection of residual starch in culture samples, 100 μl of clarified culture broth was combined with 100 μl of Lugol's iodine solution.A volume of 150 μl of the iodine-treated sample was tr ansferr ed to a tr anspar ent flatbottomed 96-well microplate and the absorbance at 580 nm was measured using microplate reader.The amount of starch was calculated using standard curves of starch.

Determination of amylolytic enzyme activity
Commercial α-amylase assays-blue and red star c h polymers T he assa ys w ere performed accor ding to the manufacturer's instructions .Briefly, co v alentl y attac hed dyes wer e liber ated fr om starc h pol ymers at a speed proportional to the α-am ylase acti vity.
The concentration of the free dyes were detected spectrophotometrically at 580 nm and converted into α-amylase activities using manufactur es calibr ation.Both substr ates ar e exclusiv el y designed for measuring α-amylase activity, as no other enzyme can act upon the substrates due to the cr oss-linka ges and the large dye molecules.
Decrease of iodine-binding star c h material according to Xiao et al. ( 2006 ) A volume of 40 μl culture supernatant was combined with 40 μl 0.1 M phosphate buffer pH 7.0 containing 0.2% gelatinized potato starch and incubated at 30 • C for 30 min.Reactions were terminated by adding 20 μl 1 M HCl.Following termination, 100 μl of Lugol's iodine solution w as added.A v olume of 150 μl of the iodinetreated sample was transferred to a transparent flat-bottomed 96well microplate and the absorbance at 580 nm was measured using a microplate reader.The amount of disappeared starch was calculated using standard curves of starch.The activity was defined as the amount of culture supernatant required for the disa ppear ance of an av er a ge of 1 μg of iodine-binding starch material per ml and minute in the assay reaction.
-v: volume of culture supernatant used (ml).
Incr ease of r educing sugar concentr ation accor ding to Miller ( 1959 ) A volume of 40 μl culture supernatant was combined with 40 μl 0.1 M phosphate buffer pH 7.0 containing 0.2% gelatinized potato starch and incubated at 30 • C for 30 min.Reactions were terminated by adding 120 μl of dinitrosalicylic acid (DNS) r ea gent and boiling reaction mixtures for 15 min at 95 • C. A volume of 150 μl of the DNS-treated sample was tr ansferr ed to a tr anspar ent flat-bottomed 96-well microplate and the absorbance at 540 nm ( A 540) was measured using a microplate reader.The activity was defined as the amount of culture supernatant required for the release of 1 μg or 1 μmol glucose per ml and minute in the assay reaction.
-v: volume of culture supernatant used (ml).

LC-MS/MS
For in-gel digestion, samples were prepared according to Lavigne et al. ( 2009 ).Briefly, the decolorization of the gel pieces was carried out in 3 × 350 μl in NH 4 HCO 3 in 50% acetonitrile in 1.5 ml Eppendorf LoBind tubes, and were incubated 30 min, gently shaken by 300 rpm at room temperature.For tryptic digestion the vacuum dried slices were treated with the Trypsin Singles Proteomics Grade Kit (Sigma-Aldrich) according to the manufacturer's instructions for in-gel digestion preparation without the reduction and alkylation step.After the incubation at 37 • C ov ernight, eac h of the samples were submerged in a new LoBind tube with 100 μl 20 mM NH 4 HCO 3 and were sonicated for 20 min.This step was carried out twice with 50 μl of 5% formic acid in 50% acetonitrile .T he samples concentr ated in the SpeedVac wer e taken up in 50 μl 0.1% formic acid and were stored at −20 • C before the next sample pr epar ation step.The Sta geTipping desalting step was carried out as described by Rappsilber et al. ( 2007 ).The tryptic peptide samples were stored at −20 • C until use for MS measurements.
For analysis of entire supernatants, protein solutions of 150 μl were mixed with 0.25 volume of 100% (w/v) TCA, incubated for 30 min on ice and the precipitated proteins were sedimented for 15 min at 16 100 g and 4 • C. The supernatants were removed and the precipitated proteins washed twice in 0.5 ml of icecold acetone.After centrifugation again (15 min, 16 100 g , 4 • C), the supernatants were discarded, the pellets were air-dried, and the pr oteins dissolv ed in 100 μl Trypsin Reaction Buffer buffer (40 mM NH 4 HCO 3 pH 8.2, 9% acetonitrile).Befor e anal yzing the protein samples with LC-MS/MS, they were digested using trypsin to cleave the proteins into peptides using the Trypsin Singles, Pr oteomics Gr ade Kit (Sigma-Aldric h) according to the manufacturer's instructions.For this, 1 μg trypsin and 1 μl of Trypsin Solubilization Reagent (contain 1 mM HCl) was added up to 100 μg Subsequent LC-MS/MS analysis of trypsin-digested samples was done as already described previously by Hünnefeld et al. ( 2021 ).
The IDA data wer e pr ocessed with ProteinPilot (5.02, Sciex) using the P ar a gon algorithm for protein identification and for building an ion library.This data was then compared with a database consisting of proteins from U. maydis 521 and U. cynodontis NBRC9727.

Plasmid cloning and strain engineering
Plasmids were constructed via Gibson assembly (Gibson et al. 2009 ) using the NEBuilder HiFi DNA Assembly Cloning Kit [New England Biolabs (NEB), Ipswich, MA, USA].The DNA oligonucleotides w ere pur chased from Eurofins Genomics (Ebersberg, Germany), and Q5 High-Fidelity DNA Polymerase (NEB) was used as the pol ymer ase.Table 2 and Table S1 provide details on the plasmids and primers used.Competent Esc heric hia coli DH5 α or PIR2 cells were used for standard cloning and plasmid maintenance, following the protocols described in Sambrook and Russell ( 2006 ).Plasmids were confirmed through polymerase chain reaction (PCR), r estriction anal ysis , or sequencing.T he protocols described in Br ac hmann et al. ( 2004) were used for the generation of pr otoplasts, tr ansformation, and isolation of genomic DNA of Ustilago strains.To integrate P etef amyA randomly into the genome, the plasmid was linearized with Bgll.For the deletion of UMAG_04064 and UMAG_02740, homologous recombination with 1000 bp flanking regions including a geneticin G418 resistance cassette were used.Successful integration and deletion were confirmed by PCR.

Genetic inventory of amylolytic enzymes encoded in Ustilago species
The genome of U. maydis 521 encodes four putative amylolytic enzymes whic h cleav e α-1,4 and α-1,6 bonds between glucose molecules (Table 3 ).Ustilago maydis 521 was taken as a r efer ence because this strain is fully sequenced and annotated (Kämper et al. 2006 ) (Refseq assembly GCF_000328475.2).A tblastn analysis (Altschul et al. 1997, Gertz et al. 2006 ) of these protein sequences against the whole-genome shotgun contigs database identified putative orthologues of all enzymes in U. cynodontis NBRC9727 with protein sequence similarities ranging between 66.2% and 87.3% (Table 3 ).Putative orthologues of all enzymes were also identified in U. maydis MB215.Inter estingl y, while high pr otein similarities were found for the α-amylase and α-glucosidase, lo w er similarities were detected for the maltase and glucoamylase.

Shake flask cultiv a tions on potato starch as a sole carbon source
In order to test the expression of these genes in vivo in axenic cultur es, cultiv ations of two pr e viousl y engineer ed itaconateov er pr oducing Ustilago str ains, U. cynodontis ITA MAX pH (Hosseinpour Tehrani et al. 2019b ) and U. maydis K14 (Becker et al. 2020 ) were performed with potato starch as a sole carbon source.To avoid the ener gy-intensiv e initial gelatinization step, the first cultiv ations wer e performed with r aw starc h po wder.Ho w e v er, no gro wth w as detected for either strain (data not shown).Consequentl y, gelatinized starc h was a pplied in the subsequent cultiv ations (Fig. 1 ).Significant differences between U. cynodontis ITA MAX pH and U. ma ydis K14 w er e observ ed in terms of growth and starc h degr adation.Whereas U. cynodontis ITA MAX pH reached an optical density of 8.9 ± 0.2 and utilized all starch and ammonium within the first 48 h (Fig. 1 A), U. ma ydis K14 sho w ed almost no growth and no decrease of starch or ammonium (Fig. 1 B), despite having the same genes present in the genome.Apparently, these genes are not expressed in U. maydis K14 under the applied conditions .T he basal expression of amylolytic enzymes may not be sufficient to sense the presence of starch.It may also be possible that there ar e differ ent triggers or e v en differ ent mec hanisms r egulating the expression of the amylolytic enzymes betw een U. ma ydis and U. cynodontis , maybe also related to pH.Howe v er, U. maydis K14 was able to grow on α-amylase pretreated starch up to OD 600 values of 5.1 ± 0.1 (Fig. 1 C), which is consistent with pr e vious liter atur e (Kr etsc hmer et al. 2017 ).

Lab-scale fermentations on gelatinized and pretrea ted pota to starch as a sole carbon source
Based on the obtained r esults, the cultiv ations wer e scaled up from shake flasks to bioreactors to analyze itaconate production on starch under more industrially relevant conditions.
In the first 72 h of the U. cynodontis ITA MAX pH fermentations on starch, the cell densities increased up to OD 600 values of ∼70 ± 1 (Fig. 2 A), and star ch w as converted to sugar mono-and maltooligomers (Fig. 2 C), which was in line with the shake flask experiments.Star ch w as mainl y hydr ol yzed to glucose with a high tr ansient accum ulation of ∼34 ± 8.6 g l −1 r eac hed after 48 h.In addition, 11.6 ± 0.6 g l −1 of maltohexaose as well as small amounts of maltose (2.3 ± 1.8 g l −1 ) were detected.Glucose and maltose were constantly metabolized until their complete depletion, whereas the concentration of maltohexaose remained almost constant until the end of the cultivation (7.8 ± 0.9 g l −1 ).This clearly indicates the inability of U. cynodontis ITA MAX pH to utilize maltohexaose.In total, this fermentation resulted in the production of 9.1 ± 1.3 g l −1 itaconate at a rate of 0.08 ± 0.01 g l −1 h −1 and a yield of 0.1 ± 0.01 g ITA g starch (unmetabolized sugars not accounted in the yield calculation).These k e y performance indicators (KPIs) ar e significantl y lo w er than the ones ac hie v ed on pur e glucose as a substrate (Hosseinpour Tehrani et al. 2019a ).Together with  2019), the r ecentl y isolated A. terreus C1 strain resulted in the production of 29.7 g l −1 itaconate at a rate of 0.21 g l −1 h −1 and a yield of 0.18 g ITA g potato starch during 3 l batch fermentation on potato starch.
In comparison to U. cynodontis ITA MAX pH, the U. maydis K14 fermentation on pr etr eated starc h accum ulated less biomass with an OD 600 of 44 after 72 h.Due to the α-amylase pr etr eatment, most of the star ch w as alr eady conv erted to sugar mono-and maltooligomers in the beginning of the fermentation, with a complete hydr ol ysis obtained after 24 h.The detected glucose concentration was lower than that during the U. cynodontis ITA MAX pH fermentation r eac hing a maxim um of 25.5 g l −1 after 24 h, whic h was fully consumed after 72 h.In contrast, higher concentrations of maltose (66.9 g l −1 ), maltotriose (42.2 g l −1 ), and maltohexaose (48.7 g l −1 ) were detected.The latter two remained almost constant until the end of the cultivation.The maltose concentration started to decline after ∼120 h, ho w e v er, a lar ge amount of this disacc haride r emained in the final culture supernatant as well.In total, this fermentation resulted in the production of 10.2 g l −1 itaconate, while the productivity was reduced to 0.05 g l −1 h −1 compared to previous fermentation of U. cynodontis ITA MAX pH due to the longer fermentation time of 192 h.The yield ac hie v ed with U. maydis K14 was slightly higher with 0.12 g ITA g starch , but 125.6 g l −1 of unmetabolized sugar (not accounted in the yield calculation) in form of maltose, maltotriose and maltohexaose remained in the culture supernatant.Hence, further optimization is needed to fully degrade accumulated maltooligomers to glucose, as this appears to be the only sugar efficiently utilized by the strains.

Analysis of the amylolytic enzyme activity in Ustilago species
To optimize the am ylolytic acti vity by metabolic engineering, enzymes present in the secretome needed to be identified and characterized.This is particularly interesting for U. cynodontis ITA MAX pH, as this strain is capable of growing on starch.The distribution of sugar mono-and maltooligomers detected during the fermentations sho w ed r elativ el y high le v els of glucose, hinting at the exoenzymatic degradation of starch by the secretion of a glucoamylase and/or α-glucosidase.Both enzymes have alr eady been fr equentl y r eported for a variety of fungi including for example A. niger , A. awamori , A. oryzae , Neurospor a cr assa , Colletotrichum gloeosporioides (Pandey et al. 2000 , Kumar andSatyanar ayana 2009 ).Contr ary, endoenzymatic tr eatment of starc h typ-  icall y r esults in accum ulation of G2 (maltose), G3 (maltotriose), G6 (maltohexaose), and G7 (maltoheptaose) maltooligomers as observed after the α-amylase treatment (Fig. 2 D), but was less pronounced during U. cynodontis ITA MAX pH fermentation.To test the hypothesis regarding the presence of a glucoamylase and/or α-glucosidase and the absence of an α-amylase in the U. cynodontis ITA MAX pH secretome, ad ditional shak e flask culti v ations wer e performed on starch, and supernatants were analyzed for their α-am ylase acti vity.Indeed, the culture supernatants did not ex-hibit any α-amylase activity, confirming the absence or very low activity of this enzyme.
Analysis of the secretome via SDS-PAGE and LC-MS/MS exposed a set of extracellular proteins, which are exclusively produced b y U. c ynodontis ITA MAX pH when grown on starch as a sole carbon source (Fig. 3 ).This confirms the enzymes as starchinduced proteins absent upon cultivation on the conventional feedstock glucose.In contrast to U. cynodontis ITA MAX pH, U. maydis K14 culture supernatants sho w ed no detectable proteins when Subsequent LC-MS/MS analysis confirmed the presence of a glucoamylase as pr e viousl y identified by blast analysis (Table 3 , Fig. 3 , Fig. S1 ), supporting the initial hypothesis.In contrast, the genome-encoded α-glucosidase (118.0 kDa) was not identified in the secretome via LC-MS/MS.Ho w ever, a w eak protein band could be observed > 130 kDa, which might reflect the expected α-glucosidase in its glycosylated form.In addition, a further enzyme belonging to the glycoside hydrolase family 93 (GH93) was detected in high abundance (Fig. 3 , Fig. S2 ).According to the Carbohydr ate-Activ e-enZYmes (C AZy) database , this enzyme shows closest similarity to an exo-α-1,5l -ar abinofur anosidase (Drula et al. 2021 ).Based on the functional description of a GH93 enzyme in the pythopathogenic fungi Fusarium graminearum , this enzyme cleaves l -arabinose side chains from arabinosesubstituted oligosaccharides with a strict substrate specificity for linear α-1,5-linked ar abinans (Car a pito et al. 2009 ).A similar enzyme activity has been reported for an exo-α-1,5l -arabinanase (GH93) from both Chrysosporium lucknowense C1 (Kühnel et al. 2011 ) and P enicillium c hrysogenum 31B (Sakamoto et al. 2004 ) r eleasing ar abinobiose fr om the nonr educing end of ar abinose oligomers.Since arabinan polymers are an important substitution of hemicellulosic and pectic oligosaccharides in plants (Yeoman et al. 2010 ), their hydr ol ysis plays an important role in the complete degradation of the plant cell wall components and is assumed to facilitate enzymatic access of the backbone (Thakur et al. 2019 ).LC-MS/MS anal ysis also r e v ealed the pr esence of an enzyme with 82.3% protein sequence similarity to UMAG_03246 (Fig. 3 , Fig. S3 ).UMAG_03246 was pr e viousl y detected in U. maydis when grown on xylan (Geiser et al. 2013 ).This enzyme is classified as part of the AA3_2 subfamily of the glucose-methanol-choline flavin-de pendent o xidor eductase famil y.The subfamil y also includes UMAG_04044, which is one of the most abundant enzymes in the secretome of U. maydis when grown on maize (Couturier et al. 2012 ).Further c har acterization identified UMAG_04044 as an aryl-alcohol oxidase (EC 1.1.3.7) with anisyl alcohol, a methoxylated lignin model compound, as the main substrate .T his suggests its functional role in lignocellulose deconstruction through lignin degr adation, pr esumabl y by pr oducing hydr ogen per oxide (Hernandez-Ortega et al. 2012, Couturier et al. 2016 ).Although these latter two secreted enzymes have no clear relation to starch degradation, a concomitant secretion of amylolytic enzymes, the exo-α-1,5l -ar abinofur anosidase as well as the aryl-alcohol oxidase in U. cynodontis ITA MAX pH on starch may be associated to its pythopathogenic lifestyle.Lignocellulose-degrading enzymes are supposed to soft or partly degrade the plant cell wall (Doehlemann et al. 2008 ), which may facilitate access to starch as the primary stor a ge carbohydr ate in plants .T he potato starch used in this study might r etain tr ace amounts of lignin and heter opol ysacc harides fr om the potato peels, triggering secretion of these putative enzymes (Rodriguez-Martinez et al. 2023 ).Additional proteins have been detected through SDS-PAGE, but could not be identified due to their low abundance .T his ma y be caused by the minimal expression of plant cell wall-degrading enzymes to pr e v ent triggering an imm une r esponse in the plants ( Doehlemann et al. 2008 ).Accordingl y, it is r easonable to assume that further cellulases and/or xylanases r equir ed for lignocellulose depol ymerization ar e pr esent in the secr etome during gr owth on starch.
Among the identified enzymes-either via LC-MS/MS in the secretome or via tblastn analysis of the U. cynodontis genomethe α-am ylase, glucoam ylase and α-glucosidase ar e typicall y r elated to starch degradation (Vihinen and Mäntsälä 1989 ).Since no α-am ylase acti vity could be detected, we deleted the glucoam ylase and α-glucosidase-encoding genes in the U. cynodontis ITA MAX pH to test their involvement in starch hydrolysis.
Remarkabl y, the knoc k out of either glucoamylase or αglucosidase resulted in a reduced, but not abolished growth and itaconate production, thus indicating a redundancy of both enzymes in starch degradation (Fig. 4 A and B).It is possible that the accumulation of limit dextrins occurs due to the pr eferr ed α-1,4 bond hydr ol ysis of the α-glucosidase (Lee et al. 2013 ).Subsequentl y, glucoamylase may efficientl y degr ade the r emaining α-1,6 linkages.Based on these in vivo results and the tblastn analysis (Table 3 ), the gene with the GenBank accession number CAKMXY010000008 (region: 881 932-885 099) encoding the αglucosidase is designated as aglA and the gene with the GenBank accession number CAKMXY010000014 (region: 83 686-85 185) encoding the glucoamylase is designated as glaA .The genes and enzymes are named according to liter atur e conv ention described in Murphy et al. ( 2011 ).Remarkably, the two highly abundant, yet unc har acterized exo-α-1,5l -ar abinofur anosidase (GenBank accession number CAKMXY010000018, region 665 749-665 919, 666 077-666 352, and 666 447-667 148) and aryl-alcohol oxidase (GenBank accession number CAKMXY010000011, region 416 218-416 544 and 416 702-418 270) do not appear to be significantly involved in starch degradation.
The amylolytic activity of the secretome was determined using gelatinized potato starch as substrate.To this end, starch degradation and reducing sugar accumulation during U. cynodontis ITA MAX pH and U. maydis K14 cultivation on starc h wer e monitor ed thr oughout the entir e cultiv ation via DNS and iodine assa ys (Fig. 5 ).T hese assa ys enable a differentiation between exoenzymes, such as glucoamylases and α-glucosidases, and endoenzymes, such as α-amylases.
The activity in the U. cynodontis ITA MAX pH cultur e incr eased almost linearly during the first 48 h, r eac hing ∼82.3 ± 5 μg min −1 ml −1 as measured by the DNS assay and 70 ± 4 μg min −1 ml −1 as measured by the iodine assay (Fig. 5 A).Nitrogen limitation after 48 h (cf.Fig. 1 A) pr e v ented further protein synthesis and led to a stabilization of the amylolytic activity.The activity of the secreted enzymes remained at its maximum level, indicating high stability.Since glucoamylases and α-glucosidases usually lead to a reduction in starch staining capacity along with a significant release of reducing sugars (Glose et al. 1990 ), the compar able activities measur ed by both methods serv e as a further confirmation for the presence of these two exoenzymes.Contrary, α-am ylase acti vity typically sho ws up b y a r a pid decr ease in iodine staining capacity with only a small amount of reducing sugars released.Ov er all, these r esults clearl y r eflect the phytopathogenic lifestyle of Ustilago species.For bitrophic growth, fungal plant degradation by CAZy needs to be restricted to a minim um le v el r equir ed for penetration (Doehlemann et al. 2008 ).Higher, unregulated activity would cause se v er e dama ge and trigger the plant immune system through sensing of plant cell wall oligomers (Wan et al. 2021 ), which can be assumed to be also the case for rapid αamylase-mediated release of starch oligomers.In contrast, exoenzymes lik e glucoam ylases and α-glucosidases were shown to primaril y r elease glucose in a more controlled fashion, thereby probabl y circumv enting str ong activ ation of the imm une r esponse.
To allow better comparison with amylolytic activities in other fungi, the volumetric enzyme activities in μmol min −1 ml −1 (U ml −1 ) were calculated based on the DNS assay using the molecular weight of glucose (Fig. 5 B).A maximum enzyme activity of 0.5 ± 0.04 U ml −1 was detected at the fifth day after inoculation of U. cynodontis ITA MAX pH, which is probably a combination of the activity of both identified exoenzyme .T his le v el of enzymatic activity is consistent with other studies indicating fungal glucoam ylase acti vities between 0.3 U ml −1 for culture supernatants (Ogundero and Osunlaja 1986 ), although much lo w er than 200 U ml −1 for commerciall y av ailable glucoamylases (Sigma-Aldric h).The activities of α-glucosidases are mostly reported as specific activities in U mg −1 , which makes the comparison with activities dir ectl y measur ed in cultur e supernatants without prior enzyme purification difficult.
The hydr ol ytic activity of the glucoamylase and α-glucosidase was also tested on additional substrates (Fig. 5 B).With 0.4 ± 0.03 U ml −1 on gelatinized corn starch and 0.4 ± 0.04 U ml −1 on gelatinized wheat flo w er, the activities w er e compar able to the one observed on potato starch.Interestingly, no activity was determined on maltose, which is one of the pr eferr ed substr ates of most fungal α-glucosidases (Manjunath et al. 1983, Chiba 1988, 1997 ).Based on substrate specificity, α-glucosidases can be classified into three main gr oups.Type-I pr efer entiall y degr ades heter ogeneous linka ges (e.g. in sucrose), while types II and III prefer entiall y hydr ol yze homogenous linka ges (e .g. in maltose , maltooligomers, and starch) with Type-III α-glucosidases being more efficient at degrading polysaccharides such as starch compared to Type-II α-glucosidases (Okuyama et al. 2016 ).Ther efor e, it a ppears that the α-glucosidase present in our culture supernatant is a Type-III α-glucosidase .T his is r elativ el y r ar e among fungal α-glucosidase, as most of them tend to hydr ol yze maltose more r a pidl y than soluble starch (Chiba 1988, Tanaka et al. 2002 ).To confirm this tendency, degradation of additional maltooligomers with v arious c hain lengths could be tested, whic h r equir es prior purification of the glucosidase to pr e v ent interfer ence with the glucoam ylase acti vity.The purified α-glucosidase could also be examined regarding its transglycosylation activity.This activity has been alr eady r eported for the α-glucosidase from Mortierella alliacea , which could use glycogen and soluble starch to transfer a glycosyl residue to ethanol, thereby producing ethyl αdglucopyranoside, a noncariogenic sweetening and flavoring agent (Tanaka et al. 2002 ).

Optimization of the amylolytic activity in U. cynodontis ITA MAX pH
The efficient hydr ol ysis of starch to glucose typically involves the synergetic action of an α-amylase and a glucoamylase.Initiall y, gelatinized starc h is liquefied to maltooligomers, whic h are then hydrolyzed to glucose by glucoamylases.Since no αamylase could be detected in U. cynodontis ITA MAX pH culture supernatants despite its genomic presence, we constitutively ov er expr essed the nati ve α-am ylase gene with the GenBank accession number CAKMXY010000005 (region: 1 296 343-1 297 953) (Fig. 6 ).
T he constitutive o verexpression of the nati ve α-am ylase gene in U. cynodontis ITA MAX pH significantl y impr ov ed the gr owth on starch (Fig. 6 A), presumably due to a more efficient starch degradation.This was accompanied by a slightly improved itaconate pr oduction compar ed to the pr ogenitor str ain (Fig. 6 B).Inter estingl y, similar KPIs were obtained on glucose and starch during shake flask cultiv ations, wher eas they significantly differed during bioreactor batch fermentations (Fig. 2 ).This may be due to variations in the C/N r atio, whic h could be optimized b y lo w cell-density and/or fed-batch fermentations in follo w-up studies.
Since nativ e expr ession of CAZy is expected to be on a low le v el to minimize plant tissue damage (Doehlemann et al. 2008 ),   conversion of starch to itaconate by U. cynodontis ITA MAX pH can likely be further optimized by constitutive overexpression of the nati ve am ylolytic genes .T his might also enable starch degradation b y U. ma ydis K14.In addition, the heter ologous ov er expr ession of α-amylases containing a starch-binding domain (Janecek et al. 2003 ) could be tested for the utilization of r aw starc h.This would eliminate the ener gy-intensiv e initial gelatinization step (Robertson et al. 2006 ) (cf.Fig. 1 ), and has already been successfull y demonstr ated for A. terreus (Wong et al. 2007 ).

Conclusion
In this w ork, w e investigated the utilization of the low-cost substr ate starc h by U. cynodontis ITA MAX pH and U. maydis K14, two Ustilago strains that have been previously engineered for efficient itaconate production.Ustilago cynodontis ITA MAX pH was able to metabolize gelatinized potato starc h, r eac hing itaconate titers of up 10 g l −1 with a yield of ∼0.1 g ITA g starch −1 during respectiv e batc h fermentations .T his could be traced back to the activity of a glucoamylase and an α-glucosidase in its secretome, whic h wer e shown to be syner gisticall y involv ed in starc h degr adation.In contrast, U. maydis K14 required α-amylase pretreated potato starch hydrolysates for growth and itaconate production.Although the KPIs are y et lo w er compared to those ac hie v ed with glucose in batch fermentations, the utilization of starch has the adv anta ge of causing less osmotic stress.While high concentrations of monomeric glucose at the start of the fermentation typicall y r esult in incr eased osmotic str ess of the deepl y engineer ed Ustilago strains, continuous enzymatic liberation of single glucose molecules from glucose polymers and their immediate consumption circumvent higher glucose accumulation.Ho w ever, to exploit the full potential of starch as a substrate for itaconate production, further optimizations such as the constitutive overexpression of the amylolytic genes or the utilization of partly in situ hydrolyzed starc h ar e r equir ed to ac hie v e higher KPIs.Although further optimization is r equir ed, U. cynodontis ITA MAX pH has been successfull y demonstr ated to be a pr omising host for itaconate pr oduction from gelatinized potato starch.

Ac kno wledgments
We thank all project partners for fruitful discussions.

Figure 1 .
Figure 1.Shake flask cultivations of U. cynodontis ITA MAX pH and U. maydis K14 on gelatinized potato starch (A and B) and α-amylase pretreated potato starch (C) as a sole carbon source.Shake flask cultiv ations wer e performed in MTM medium containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and 10 g l −1 gelatinized potato starch.The mean values with standard deviation of two independent biological replicates are shown.

Figure 2 .
Figure 2. High-density batch fermentations on gelatinized potato starch (A and C) and α-amylase pretreated potato starch (B and D) as a sole carbon source.Concentration of potato starch ( ), maltohexaose ( ), maltotriose ( ), maltose ( ), glucose ( ), OD 600 ( ), ammonium ( ), and itaconate ( ) during fermentation in a bioreactor containing batch medium with 75 mM NH 4 Cl and either 100 g l −1 gelatinized potato starch (A and C) or 200 g l −1 α-amylase pr etr eated potato starc h (B and D).The pH was contr olled at pH 3.6 by automatic titr ation with 5 M NaOH.The mean v alues with standard deviation of two independent biological replicates are shown for (A) and (C), while (B) and (D) show the values of a single re presentati ve culture.

Figure 3 .
Figure 3. SDS-PAGE and LC-MS/MS analysis of U. cynodontis ITA MAX pH and U. maydis K14 culture supernatants.Shake flask cultivations were performed in MTM medium containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and either 10 g l −1 gelatinized potato starch or 10 g l −1 glucose.Culture supernatants were 40x concentrated using 10 kDa MWCO spin columns and afterw ar ds separated electrophoretically using a 12% polyacrylamide gel.Two bands (indicated by the red arrows) were excised, trypsin-digested, and subjected to LC-MS/MS analysis .T he entire supernatant samples were also analyzed via LC-MS/MS.The predicted signal peptide sequences as indicated in Figs S1 -S3 are not accounted in the % coverage (95%) values.

Figure 4 .
Figure 4. Shake flask cultivations of U. cynodontis ITA MAX pH deletion mutants on gelatinized potato starch as a sole carbon source.Shake flask cultiv ations wer e performed in MTM medium containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and 50 g l −1 gelatinized potato starch.All modifications were performed in U. cynodontis ITA MAX pH.(A) Optical densities and (B) itaconate concentrations throughout the cultivations are shown.The values of a single r epr esentativ e cultur e ar e shown.

Figure 5 .
Figure5.Am ylolytic acti vity in supernatants of U. cynodontis ITA MAX pH and U. ma ydis K14 gro wn on gelatinized potato star ch as a sole carbon sour ce.Shake flask cultivations were performed in MTM medium containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and 10 g l −1 gelatinized potato starch.(A) Am ylolytic acti vity in μg min −1 ml −1 during the cultiv ation, quantified via DNS (blac k line) and iodine assay (or ange line).(B) Volumetric enzyme activities in U ml −1 on potato star ch, corn star ch, wheat flour, and maltose, calculated based on the molecular weight of glucose .T he mean values with standard deviation of three independent biological replicates are shown.ND: not detected;/: not analyzed due to technical limitations like viscosity.

Figure 6 .
Figure 6.Shake flask cultivations of U. cynodontis ITA MAX pH constitutiv el y expr essing the nativ e α-amylase gene on gelatinized potato starc h as a sole carbon source.Shake flask cultivations were performed in MTM medium containing 15 mM NH 4 Cl, 100 mM MES pH 6.5, and 50 g l −1 gelatinized potato starch.(A) Optical densities and (B) itaconate concentrations throughout the cultivations are shown.The values of a single re presentati ve cultur e ar e shown.The OD 600 v alues and itaconate concentr ations of U. cynodontis ITA MAX pH fr om Fig. 4 ar e shown a gain for comparison.In addition, values form a previous cultivation of U. cynodontis ITA MAX pH on 50 g l −1 glucose are shown. 4

Table 1 .
Ustilago strains used in this study.

Table 2 .
Plasmids used in this study.

Table 3 .
(Huang et al. 2014resent in U. maydis due to unmetabolized maltohexaose, this clearly emphasizes the need of optimization, for instance by ov er pr oduction of additional amylol ytic enzymes.Suc h an a ppr oac h was alr eady successfull y a pplied for pr oduction of itaconate fr om liquefied corn starch with A. terreus CICC 40205.While the wildtype str ain pr oduced ∼14 g l −1 itaconate, heter ologous ov er expr ession of a glucoamylase gene from Aspergillus niger increased production to ∼60 g l −1(Huang et al. 2014).